Evolutionarily Conserved Fragments reveal modular architecture and expand functional coverage in bacteriophage proteins

Abstract

Bacteriophage proteins evolve through extensive modular exchange, yet the sequence units underlying this process remain poorly defined. Classical domains are plausible evolutionary modules, but they incompletely annotate phage proteins and may not always coincide with the regions conserved across protein families. Here, we constructed 13,035 Evolutionarily Conserved Fragments (ECFs), grouped into 3,167 families, through large-scale HMM-HMM comparisons of proteins from 5,396 RefSeq phage genomes, without imposing predefined domain boundaries. ECFs overlapped substantially with ECOD and Pfam annotations but formed a heterogeneous spectrum comprising subdomains, domain-equivalent units, composite regions and fragments with no counterpart in either resource. Their relationships were frequently nested, indicating that conserved modules occur at multiple organisational scales. ECFs annotated 50% of phage representative proteins (at least doubling the coverage of either database), with the largest gains in structural and infection-related functions, a pattern replicated in independent metagenomic data. ECF families also showed function-dependent patterns of diversity and spatial segregation and provided functional associations for 23,925 protein families lacking detectable Pfam or ECOD domains. In receptor-binding proteins, ECFs subdivided established domains, delineated previously unannotated structural elements and recovered homologous regions where structural similarity was ambiguous. These results show that the evolutionary building blocks of phage proteins do not always coincide with classical domains, highlighting that evolutionary processes act across multiple scales. ECFs provide a complementary, sequence-derived framework for investigating how complex viral proteins are assembled, diversified and functionally organised.